ABSTRACT
The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.
Subject(s)
Freezing , Vitamin D/analogs & derivatives , Vitamin D/blood , Chromatography, Liquid/methods , Humans , Tandem Mass Spectrometry/methodsABSTRACT
An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.
Subject(s)
Tandem Mass Spectrometry , Vitamin D , 25-Hydroxyvitamin D 2 , Chromatography, Liquid/methods , Reference Standards , Tandem Mass Spectrometry/methods , Vitamin D/analogs & derivativesABSTRACT
An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.
Subject(s)
Societies, Medical/standards , Vitamin D/analogs & derivatives , Vitamin D/chemistry , Humans , Reference Standards , Specimen Handling , Vitamin D/bloodABSTRACT
OBJECTIVE: The aim of the study was to determine the range of serum sex-related steroids in normal postmenopausal women and in women of the same age with a diagnosis of vulvovaginal atrophy (VVA). METHODS: Validated mass spectrometry-based assays coupled to gas or liquid chromatography were used over a 10-year period for steroid measurements. Serum samples were obtained in up to 1,512 women aged 55 to 65 years. RESULTS: Serum estrone sulfate (E1S) and androsterone glucuronide (ADT-G), the main metabolites of estrogens and androgens, respectively, were 16.9% (Pâ=â0.005) and 16.1% (Pâ=â0.001) higher in women not diagnosed with moderate/severe VVA than those diagnosed with VVA. Serum estrone (E1) was 14.5% (Pâ<â0.0001) higher in women with no diagnosis of VVA, whereas the other steroids did not show meaningful differences. The limited biological significance of serum estradiol (E2) and testosterone is supported by the lack of statistical significance in the serum concentrations of these two steroids between the two groups. Most importantly, for the women without a diagnosis of VVA, the normal upper limit (95 centile) of serum E2 was 9.15 pg/mL (nâ=â364) and 10.7 pg/mL (nâ=â67) for a weighted average of 9.99 pg E2/mL. A limit of 10 pg E2/mL has recently been found by two other laboratories. When comparing 50- to 59-year-old and 70- to 79-year-old women, serum E2, E1S, ADT-G, and DHEA were, respectively, 24.4%, 22.6%, 27.0%, and 85.9% higher in the younger group. CONCLUSIONS: Somewhat higher values, namely, 16.9% and 16.1%, are observed in the serum concentrations of the estrogen (E1S) and androgen (ADT-G) metabolites in normal compared with women with a diagnosis of VVA. Such data indicating a lower estrogenic and androgenic global exposure in women diagnosed with VVA offers an opportunity for the local intravaginal administration of DHEA to replace the deficiency in endogenous DHEA.
Subject(s)
Androsterone/analogs & derivatives , Atrophy , Estrone/analogs & derivatives , Postmenopause/blood , Vaginal Diseases/blood , Aged , Androsterone/blood , Case-Control Studies , Clinical Trials as Topic , Dehydroepiandrosterone Sulfate/blood , Estrone/blood , Female , Humans , Mass Spectrometry , Middle Aged , Testosterone/bloodABSTRACT
In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315>97 & 315>109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315>97-315>109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315>109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1×3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70%â¼80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1×3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5-2000pg/mL, a good linearity is observed with R>0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74â¼91.7pg/mL, which is well below the reported concentrations of 314 pg/mLâ¼942pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mLâ¼18.6ng/mL.
Subject(s)
Chromatography, High Pressure Liquid/methods , Progesterone/blood , Progesterone/isolation & purification , Tandem Mass Spectrometry/methods , Female , Humans , Limit of Detection , Liquid-Liquid Extraction/methods , Postmenopause/blood , Premenopause/bloodABSTRACT
The concentrations of allopregnanolone (Allopreg), pregnenolone (Preg) and androsterone (ADT) are very low in the circulation, especially in postmenopausal women, resulting in a considerable challenge for their accurate measurements in serum or plasma. In this report, a sensitive and reliable LC-MS/MS assay method has been developed using a simple sample preparation and the 1-Amino-4-methylpiperazine (AMP) derivatization procedure. A 5pg/ml (0.1pg on column) of low limit of quantitation has been achieved for Allopreg, Preg and ADT, with a sensitivity comparable to data obtained with the commercial reagent. The major benefit of this reagent is to limit the matrix effect since the excess amount of reagent can be removed during the reaction. Multiple reaction monitoring (MRM) from the derivatization of AMP not only increases the detection of these compounds but also provides a good resolution for Allopreg, Preg and ADT from interferences, especially for Allopreg from its isomers. Within the calibration range of 5pg/ml to 2000pg/ml, a good linearity was obtained with R>0.99 where the weighing factor is 1/X. Bias and coefficients of variance are within 15% for all QC levels. The matrix effect has been evaluated, well meeting the acceptance criteria according to the FDA guidelines. With this method, the concentrations of Allopreg, Preg and ADT in postmenopausal serum are in the range of 6.4-53.6pg/ml, 16.2-68.0pg/ml and 23.9-114.0pg/ml, respectively, while the ranges in premenopausal serum are 8.2-701.5pg/ml, 31.2-135.2pg/ml and 47.8-310.0pg/ml, respectively.
Subject(s)
Androsterone/blood , Chromatography, Liquid/methods , Piperazines/chemistry , Postmenopause/blood , Pregnanolone/blood , Pregnenolone/blood , Premenopause/blood , Tandem Mass Spectrometry/methods , Androsterone/chemistry , Biological Assay/methods , Female , Humans , Molecular Structure , Pregnanolone/chemistry , Pregnenolone/chemistry , Reproducibility of ResultsABSTRACT
CONTEXT: Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. OBJECTIVE: To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. PARTICIPANTS: Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). OUTCOMES: Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. SETTING: Academic medical center. RESULTS: Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17ß-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. CONCLUSIONS: We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression.
Subject(s)
5-alpha Reductase Inhibitors/adverse effects , Alopecia/drug therapy , Androgens/blood , Depressive Disorder, Major , Finasteride/adverse effects , Receptors, Androgen/metabolism , Sexual Dysfunctions, Psychological , Adult , Depressive Disorder, Major/blood , Depressive Disorder, Major/chemically induced , Depressive Disorder, Major/physiopathology , Gene Expression , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Sexual Dysfunction, Physiological/blood , Sexual Dysfunction, Physiological/chemically induced , Sexual Dysfunction, Physiological/physiopathology , Sexual Dysfunctions, Psychological/blood , Sexual Dysfunctions, Psychological/chemically induced , Sexual Dysfunctions, Psychological/physiopathology , Young AdultABSTRACT
This study integrates all data obtained in women aged 40-80years enrolled with moderate to severe symptoms of vulvovaginal atrophy (VVA) who received daily intravaginal administration of 0.50% (6.5mg) dehydroepiandrosterone (DHEA; prasterone) for 12weeks (n=723; ITT-S population) as compared with placebo (n=266; ITT-S population). To this end, serum steroid levels (DHEA, DHEA-sulfate (DHEA-S), androst-5-ene-3ß, 17ß-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17ß-diol 17-glucuronide (3α-diol-17G)) were measured at Day 1 and Week 12 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following validation performed according to the FDA guidelines [1-6]. In agreement with the mechanisms of intracrinology where DHEA is exclusively transformed intracellularly into active sex steroids which act and are inactivated locally before being released as glucuronided or sulfated metabolites for elimination by the kidneys and liver, all sex steroids remained well within normal postmenopausal values following administration of intravaginal DHEA. Serum estradiol, the most relevant sex steroid, was measured after 12weeks of treatment at 3.36pg/ml (cITT-S population) or 19% below the normal postmenopausal value of 4.17pg/ml. On the other hand, serum E1-S, the best recognized marker of global estrogenic activity, shows an average value of 209pg/ml at 12 weeks compared to 220pg/ml in normal postmenopausal women. Moreover, serum ADT-G, the main metabolite of androgens, also remains well within normal postmenopausal values. The present data shows that a low daily intravaginal dose (6.5mg) of DHEA (prasterone) which is efficacious on the symptoms and signs of VVA, permits to achieve the desired local efficacy without systemic exposure, in agreement with the stringent mechanisms of menopause established after 500 million years of evolution where each cell in each tissue is the master of its sex steroid exposure.
Subject(s)
Dehydroepiandrosterone/administration & dosage , Estradiol/blood , Postmenopause/blood , Testosterone/blood , Vaginal Diseases/drug therapy , Administration, Intravaginal , Adult , Aged , Aged, 80 and over , Atrophy/drug therapy , Dehydroepiandrosterone/pharmacokinetics , Female , Hormone Replacement Therapy , Humans , Middle AgedABSTRACT
7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7ß OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC-MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10 pg/mL (0.2 pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10-2000 pg/mL, a good linearity was obtained with R>0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze-thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7ß OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7ß OH-DHEA in both types of sera.
Subject(s)
Blood Chemical Analysis/methods , Chromatography, Liquid/methods , Dehydroepiandrosterone/blood , Dehydroepiandrosterone/chemistry , Limit of Detection , Tandem Mass Spectrometry/methods , Adult , Calibration , Dehydroepiandrosterone/pharmacology , Female , Hemolysis/drug effects , Humans , Hyperlipidemias/chemically induced , Indicators and Reagents/chemistry , Linear Models , Middle Aged , Postmenopause/blood , Premenopause/blood , Young AdultABSTRACT
The circulating levels of 16ß hydroxydehydroepiandrosterone (16ß OH-DHEA) are at the limit of detection (less than 10 pg/mL), unlike the serum concentrations of 16α-hydroxydehydroepiandrosterone (16α OH-DHEA, 10-300 pg/mL) in premenopausal, postmenopausal and male serum. A major reason could be the rapid conversion of 16ß OH-DHEA to 5-androstene-3beta, 17beta-diol 16 one (3ß, 17ß-diol 16-oxo) in serum due to the stereospecific structure of 16ß OH-DHEA. In ultrapure H2O, there is no apparent conversion observed while 16ß OH-DHEA (10 ng/mL) spiked in stripped or unstripped serum is quickly converted to 3ß, 17ß-diol 16-oxo at room temperature. During this conversion, a further converted product was observed with a difference in molecular weight of 16 Da from that of 16ß OH-DHEA and 3ß, 17ß-diol 16-oxo, which could be their hydroxylation product, i.e. triol-ketone. Under basic conditions, further conversion occurs. The present data can explain the practically undetectable concentration of serum 16ß OH-DHEA while 3ß, 17ß-diol 16-oxo is at the level of less than 50 pg/mL. Serum concentrations of (0.0-9.9 pg/mL for 16ß OH-DHEA, 8.9-50.7 pg/mL for 3ß, 17ß-diol 16-oxo and 10.0-285.0 pg/mL for 16α OH-DHEA are measured in sera of premenopausal, postmenopausal women and men over 50 years of age.
Subject(s)
Blood Chemical Analysis/methods , Dehydroepiandrosterone/analogs & derivatives , Adult , Dehydroepiandrosterone/blood , Dehydroepiandrosterone/chemistry , Female , Humans , Ketones/chemistry , Limit of Detection , Male , Middle Aged , Young AdultABSTRACT
OBJECTIVE: Analyze the serum levels of DHEA (prasterone) and its metabolites after daily intravaginal 0.50% (6.5 mg) DHEA in postmenopausal women with vulvovaginal atrophy (VVA). METHODS: Serum samples were obtained at baseline and after 12, 26 and 52 weeks of treatment. The serum levels of DHEA, DHEA-sulfate (DHEA-S), androstene-3ß, 17ß-diol (5-diol), androstenedione (4-dione), testosterone, dihydrotestosterone (DHT), estrone (E1), estradiol (E2), E1-sulfate (E1-S), androsterone glucuronide (ADT-G) and androstane-3α,17ß-diol 17-glucuronide (3α-diol-17G) were measured by validated liquid chromatography-tandem mass spectrometry. RESULTS: A total of 435 women were exposed for 52 weeks. All serum steroids remained within normal values with no significant differences between lengths of treatment. For the most relevant estrogen-related compounds, namely E1, E2, and E1-S, a reliable marker of total estrogen exposure, the values in the DHEA-treated group at 52 weeks were -3.4%, -9.1% and +1.8%, respectively, compared to the normal postmenopausal values, thus clearly confirming the absence of significant systemic estrogen exposure. CONCLUSION: While confirming that all serum sex steroids originating exclusively from DHEA after menopause are maintained within the normal postmenopausal values, the present data show that the dose of intravaginal DHEA used is free from systemic exposure with no detectable change in metabolism up to 52 weeks of treatment.
Subject(s)
Dehydroepiandrosterone/administration & dosage , Gonadal Steroid Hormones/blood , Administration, Intravaginal , Adult , Aged , Dehydroepiandrosterone/blood , Dehydroepiandrosterone Sulfate/blood , Estradiol/blood , Female , Humans , Middle Aged , Postmenopause , Reference Values , Testosterone/bloodABSTRACT
The objective of the present phase III, placebo-controlled, double-blind, prospective and randomized study was to confirm the efficacy of daily intravaginal administration of 0.50% dehydroepiandrosterone (DHEA; prasterone) ovules for 12 weeks on moderate to severe dyspareunia (or pain at sexual activity) as most bothersome symptom of vulvovaginal atrophy (VVA) while having serum steroid concentrations within normal postmenopausal values. To this end, serum levels of DHEA, DHEA-sulfate (DHEA-S), Androst-5-ene-diol-3ß, 17ß-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17ß-diol 17-glucuronide (3α-diol-17G) were measured by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). In agreement with the mechanisms of intracrinology, all serum sex steroids and metabolites concentrations after 12 weeks of daily intravaginal administration of 0.50% DHEA remain well within the limits of normal postmenopausal women. More specifically, the 12-week serum E2 concentration was measured at 22% below the average normal postmenopausal value (3.26 versus 4.17 pg/ml), thus eliminating any fear of E2 exposure outside the vagina. In addition, serum E1-S, a particularly reliable indicator of global estrogenic activity, shows serum levels practically superimposable to the value observed in normal postmenopausal women (219 versus 220 pg/ml). Similarly, serum ADT-G, the major metabolite of androgens, remains within normal postmenopausal values. The present data confirm the intracellular transformation of DHEA in the vagina resulting in local efficacy without any systemic exposure to sex steroids, observations which are in agreement with the physiological mechanisms of menopause.
Subject(s)
Dehydroepiandrosterone/administration & dosage , Gonadal Steroid Hormones/blood , Vagina , Aged , Double-Blind Method , Drug Administration Routes , Female , Humans , Middle Aged , Placebos , Postmenopause , Prospective StudiesSubject(s)
Chromatography, Liquid , Estrogens/blood , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Chromatography, Liquid/standards , Female , Humans , Menopause/blood , Reference Standards , Tandem Mass Spectrometry/methods , Tandem Mass Spectrometry/standards , United States , United States Food and Drug Administration , Validation Studies as TopicABSTRACT
Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200µL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.
Subject(s)
Androstane-3,17-diol/analogs & derivatives , Androsterone/analogs & derivatives , Chromatography, High Pressure Liquid/methods , Postmenopause/blood , Tandem Mass Spectrometry/methods , Androstane-3,17-diol/blood , Androsterone/blood , Chromatography, High Pressure Liquid/economics , Female , Humans , Limit of Detection , Tandem Mass Spectrometry/economicsABSTRACT
Conventionally, the concentration of steroidal sulfates was estimated by indirect or immunobased assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1S), androsterone sulfate (ADTS), pregnenolone sulfate (PregS) and allopregnanolone sulfate (AllopregS). E1S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40ng/mL, 40pg/mL, 5ng/mL, 1.5ng/mL and 0.25ng/mL for DHEAS, E1-S, ADTS, PregS and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1S, ADTS and PregS while for AllopregS, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines.
Subject(s)
Androsterone/analogs & derivatives , Dehydroepiandrosterone Sulfate/blood , Estrone/analogs & derivatives , Pregnanolone/blood , Pregnenolone/blood , Tandem Mass Spectrometry/methods , Androsterone/blood , Chromatography, Liquid/methods , Estrone/blood , Humans , Limit of Detection , Reproducibility of ResultsABSTRACT
Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC-MS or GC-MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3ß, 17ß-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC-MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid-liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1pg/mL, 4pg/mL, 50pg/mL, 10pg/mL, 100pg/mL, 500pg/mL and 100pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4pg/mL with S/N≥5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1-84.7% for E2, 83.6-94.5% for E1, 88.2-90.3% for Testo, 82.0-90.6% for DHT, 84.9-92.0% for 5-diol, 88.1-93.8% for DHEA and 86.2-90.3% for 4-dione, respectively. A good linearity is obtained with R>0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1-8.9% for the low limits of quantification of all seven compounds with 0.7-6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC-MS/MS and LC-MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.